Chimeric antigen receptor to target trop-2-positive cancers

Abstract

Embodiments of the disclosure include methods and compositions related to targeting of TROP-2-expressing cells with particular engineered receptors. In specific embodiments, NK cells are specifically engineered to bind TROP-2 using particular chimeric antigen receptor constructs. In certain embodiments, vectors that express the TROP-2-targeting CARs also express a particular suicide gene and/or one or more particular cytokines.

What is claimed is: 
     
         1 . A polynucleotide that encodes an anti-TROP-2 chimeric antigen receptor (CAR), the CAR comprising (i) an anti-TROP-2 antigen binding region of a TROP-2 specific antibody, (ii) a transmembrane domain, and (iii) an intracellular region. 
     
     
         2 . The polynucleotide of  claim 1 , wherein the TROP-2 specific antibody is an RS7 antibody. 
     
     
         3 . The polynucleotide of  claim 2 , wherein the RS7 antibody is murine RS7 (mRS7). 
     
     
         4 . The polynucleotide of  claim 2 , wherein the RS7 antibody is humanized RS7 (hRS7). 
     
     
         5 . The polynucleotide of any of  claims 1-4 , wherein the anti-TROP-2 antigen binding region comprises a sequence having at least 85% sequence identity with SEQ ID NO:9. 
     
     
         6 . The polynucleotide of any of  claims 1-5 , wherein the anti-TROP-2 antigen binding region comprises a sequence having at least 85% sequence identity with SEQ ID NO:14. 
     
     
         7 . The polynucleotide of any of  claims 1-6 , wherein the anti-TROP-2 antigen binding region comprises a first sequence having at least 85% sequence identity with SEQ ID NO:9 and a second sequence having at least 85% sequence identity with SEQ ID NO:14. 
     
     
         8 . The polynucleotide of any of  claims 1-7 , wherein the anti-TROP-2 antigen binding region comprises SEQ ID NO:9. 
     
     
         9 . The polynucleotide of any of  claims 1-8 , wherein the anti-TROP-2 antigen binding region comprises SEQ ID NO:14. 
     
     
         10 . The polynucleotide of any of  claims 1-9 , wherein the anti-TROP-2 antigen binding region comprises SEQ ID NO:9 and SEQ ID NO:14 
     
     
         11 . The polynucleotide of  claim 1 , wherein the TROP-2 specific antibody is a 2G10 antibody 
     
     
         12 . The polynucleotide of  claim 11 , wherein the 2G10 antibody is murine 2G10 (m2G10). 
     
     
         13 . The polynucleotide of  claim 11 , wherein the 2G10 antibody is human 2G10 (h2G10). 
     
     
         14 . The polynucleotide of any of  claims 11-13 , wherein the anti-TROP-2 antigen binding region comprises SEQ ID NO:44. 
     
     
         15 . The polynucleotide of any of  claims 11-13 , wherein the anti-TROP-2 antigen binding region comprises SEQ ID NO:46. 
     
     
         16 . The polynucleotide of any of  claims 1-15 , wherein the anti-TROP-2 antigen binding region is a codon optimized anti-TROP-2 antigen binding region. 
     
     
         17 . The polynucleotide of any of  claims 1-16 , wherein the transmembrane domain is a transmembrane domain from CD28, the alpha chain of the T-cell receptor, beta chain of the T-cell receptor, zeta chain of the T-cell receptor, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, DAP10, orDAP12. 
     
     
         18 . The polynucleotide of  claim 17 , wherein the transmembrane domain is a CD27 transmembrane domain. 
     
     
         19 . The polynucleotide of  claim 18 , wherein the transmembrane domain comprises SEQ ID NO:22. 
     
     
         20 . The polynucleotide of  claim 17 , wherein the transmembrane domain is a CD28 transmembrane domain. 
     
     
         21 . The polynucleotide of  claim 20 , wherein the transmembrane domain comprises SEQ ID NO:23. 
     
     
         22 . The polynucleotide of  claim 17 , wherein the transmembrane domain is a CD8 transmembrane domain. 
     
     
         23 . The polynucleotide of  claim 22 , wherein the transmembrane domain comprises SEQ ID NO:24. 
     
     
         24 . The polynucleotide of any of  claims 1-23 , wherein the intracellular domain is an intracellular domain from CD3 zeta, CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, or NKp80. 
     
     
         25 . The polynucleotide of  claim 24 , wherein the intracellular domain is a CD3 zeta intracellular domain. 
     
     
         26 . The polynucleotide of  claim 25 , wherein the intracellular domain comprises SEQ ID NO:29. 
     
     
         27 . The polynucleotide of  claim 24 , wherein the intracellular domain is a CD28 intracellular domain. 
     
     
         28 . The polynucleotide of any of  claims 1-26 , wherein the CAR comprises two or more intracellular domains. 
     
     
         29 . The polynucleotide of  claim 28 , wherein the two or more intracellular domains comprise a CD3 zeta intracellular domain and an additional intracellular domain selected from a CD28, DAP10, DAP12, 4-1BB, NKG2D, and 2B4 intracellular domain. 
     
     
         30 . The polynucleotide of  claim 28 or 29 , wherein the two or more intracellular domains comprise a CD3 zeta intracellular domain and a CD28 intracellular domain. 
     
     
         31 . The polynucleotide of  claim 28 or 29 , wherein the two or more intracellular domains comprise a CD3 zeta intracellular domain and a DAP10 intracellular domain. 
     
     
         32 . The polynucleotide of any of  claims 1-31 , further comprising a signal peptide. 
     
     
         33 . The polynucleotide of  claim 32 , wherein the signal peptide is a signal peptide from CD8, CD27, granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R), Ig heavy chain (IgH), CD3, or CD4. 
     
     
         34 . The polynucleotide of  claim 33 , wherein the signal peptide is a GMSCF-R signal peptide. 
     
     
         35 . The polynucleotide of  claim 33 or 34 , wherein the signal peptide comprises SEQ ID NO:21. 
     
     
         36 . The polynucleotide of  claim 33 , wherein the signal peptide is a IgH signal peptide. 
     
     
         37 . The polynucleotide of  claim 36 , wherein the signal peptide comprises SEQ ID NO:20. 
     
     
         38 . The polynucleotide of any of  claims 1-37 , wherein the CAR comprises an IgH signal peptide, an anti-TROP-2 antigen binding region of an RS7 antibody, a CD28 transmembrane domain, and a CD3 zeta intracellular domain. 
     
     
         39 . The polynucleotide of any of  claims 1-38 , wherein the polynucleotide further encodes an additional polypeptide of interest. 
     
     
         40 . The polynucleotide of  claim 39 , wherein the sequence encoding the additional polypeptide of interest and the sequence encoding the CAR are separated on the polynucleotide by a 2A element. 
     
     
         41 . The polynucleotide of  claim 40 , wherein the 2A element is an E2A element. 
     
     
         42 . The polynucleotide of  claim 41 , wherein the E2A element comprises SEQ ID NO:38. 
     
     
         43 . The polynucleotide of any of  claims 39-42 , wherein the additional polypeptide of interest is a therapeutic protein or a protein that enhances cell activity, expansion, and/or persistence. 
     
     
         44 . The polynucleotide of any of  claims 39-43 , wherein the additional polypeptide of interest is a suicide gene product, a cytokine, or a human or viral protein that enhances proliferation, expansion and/or metabolic fitness. 
     
     
         45 . The polynucleotide of any of  claims 39-44 , wherein the additional polypeptide of interest is a cytokine. 
     
     
         46 . The polynucleotide of  claim 45 , wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7. 
     
     
         47 . The polynucleotide of  claim 46 , wherein the cytokine is IL-15. 
     
     
         48 . The polynucleotide of  claim 47 , wherein the cytokine comprises SEQ ID NO:37. 
     
     
         49 . The polynucleotide of any of  claims 39-48 , wherein the additional polypeptide of interest is a suicide gene product. 
     
     
         50 . The polynucleotide of  claim 49 , wherein the suicide gene product is Caspase 9. 
     
     
         51 . The polynucleotide of  claim 49 or 50 , wherein the polynucleotide encodes for a sequence having at least 95% identity with SEQ ID NO:42. 
     
     
         52 . The polynucleotide of  claim 51 , wherein the polynucleotide encodes for SEQ ID NO:42. 
     
     
         53 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:2. 
     
     
         54 . The polynucleotide of  claim 53 , wherein the polynucleotide encodes for SEQ ID NO:2. 
     
     
         55 . The polynucleotide of  claim 54 , wherein the polynucleotide comprises SEQ ID NO:1. 
     
     
         56 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:4. 
     
     
         57 . The polynucleotide of  claim 56 , wherein the polynucleotide encodes for SEQ ID NO:4. 
     
     
         58 . The polynucleotide of  claim 57 , wherein the polynucleotide comprises SEQ ID NO:3. 
     
     
         59 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:6. 
     
     
         60 . The polynucleotide of  claim 59 , wherein the polynucleotide encodes for SEQ ID NO:6. 
     
     
         61 . The polynucleotide of  claim 60 , wherein the polynucleotide comprises SEQ ID NO:5. 
     
     
         62 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:8. 
     
     
         63 . The polynucleotide of  claim 62 , wherein the polynucleotide encodes for SEQ ID NO:8. 
     
     
         64 . The polynucleotide of  claim 63 , wherein the polynucleotide comprises SEQ ID NO:7. 
     
     
         65 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:51. 
     
     
         66 . The polynucleotide of  claim 65 , wherein the polynucleotide encodes for SEQ ID NO:51. 
     
     
         67 . The polynucleotide of  claim 66 , wherein the polynucleotide comprises SEQ ID NO:50. 
     
     
         68 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:53. 
     
     
         69 . The polynucleotide of  claim 68 , wherein the polynucleotide encodes for SEQ ID NO:53. 
     
     
         70 . The polynucleotide of claim  70 , wherein the polynucleotide comprises SEQ ID NO:52. 
     
     
         71 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:55. 
     
     
         72 . The polynucleotide of  claim 71 , wherein the polynucleotide encodes for SEQ ID NO:55. 
     
     
         73 . The polynucleotide of  claim 72 , wherein the polynucleotide comprises SEQ ID NO:54. 
     
     
         74 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:57. 
     
     
         75 . The polynucleotide of  claim 74 , wherein the polynucleotide encodes for SEQ ID NO:57. 
     
     
         76 . The polynucleotide of  claim 75 , wherein the polynucleotide comprises SEQ ID NO:56. 
     
     
         77 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:59. 
     
     
         78 . The polynucleotide of  claim 77 , wherein the polynucleotide encodes for SEQ ID NO:59. 
     
     
         79 . The polynucleotide of  claim 78 , wherein the polynucleotide comprises SEQ ID NO:58. 
     
     
         80 . The polynucleotide of any of  claims 1-52 , wherein the polynucleotide encodes for a sequence having at least 95% sequence identity with SEQ ID NO:61. 
     
     
         81 . The polynucleotide of  claim 80 , wherein the polynucleotide encodes for SEQ ID NO:61. 
     
     
         82 . The polynucleotide of  claim 81 , wherein the polynucleotide comprises SEQ ID NO:60. 
     
     
         83 . A vector comprising the polynucleotide of any one of  claims 1-82 . 
     
     
         84 . The vector of  claim 83 , wherein the vector is a viral vector. 
     
     
         85 . The vector of  claim 84 , wherein the viral vector is an adenoviral vector, adeno-associated viral vector, lentiviral vector, or retroviral vector. 
     
     
         86 . The vector of  claim 83 , wherein the vector is a non-viral vector. 
     
     
         87 . The vector of  claim 86 , wherein the non-viral vector is a plasmid. 
     
     
         88 . An immune cell comprising the polynucleotide of any one of  claims 1-55  or the vector of any one of  claims 83-87 . 
     
     
         89 . The immune cell of  claim 88 , wherein the immune cell is a natural killer (NK) cell, T cell, gamma delta T cell, alpha beta T cell, invariant NKT (iNKT) cell, B cell, macrophage, mesenchymal stromal cell, or dendritic cell. 
     
     
         90 . The immune cell of  claim 89 , wherein the immune cell is a NK cell 
     
     
         91 . The immune cell of  claim 90 , wherein the NK cell is derived from cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, or from a cell line. 
     
     
         92 . The immune cell of  claim 91 , wherein the NK cell is derived from a cell line, wherein the NK cell line is NK-92. 
     
     
         93 . The immune cell of  claim 91 , wherein the NK cell is derived from a cord blood mononuclear cell. 
     
     
         94 . The immune cell of any of  claims 90-93 , wherein the NK cell is a CD56+NK cell. 
     
     
         95 . The immune cell of any of  claims 90-94 , wherein the NK cell expresses a recombinant cytokine. 
     
     
         96 . The immune cell of  claim 95 , wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or IL-23. 
     
     
         97 . The immune cell of  claim 96 , wherein the cytokine is IL-15. 
     
     
         98 . The immune cell of any of  claims 88-97 , wherein expression of one or more endogenous genes in the immune cell has been modified. 
     
     
         99 . The immune cell of  claim 98 , wherein the one or more genes comprise NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38, or a combination thereof. 
     
     
         100 . A population of immune cells comprising the immune cell of any one of  claims 88-99 . 
     
     
         101 . A method of killing TROP-2-positive cells in an individual, comprising administering to the individual an effective amount of cells harboring the polynucleotide of any one of  claims 1-55 . 
     
     
         102 . The method of  claim 101 , wherein the cells harboring the polynucleotide are immune cells. 
     
     
         103 . The method of  claim 102 , wherein the immune cells are NK cells, T cells, gamma delta T cells, alpha beta T cells, invariant NKT (iNKT) cells, B cells, macrophages, dendritic cells, or a mixture thereof. 
     
     
         104 . The method of  claim 103 , wherein the immune cells comprise NK cells, wherein the NK cells are derived from cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, from a cell line, or a mixture thereof. 
     
     
         105 . The method of  claim 104 , wherein the NK cells are derived from cord blood mononuclear cells. 
     
     
         106 . The method of any of  claims 102-105 , wherein the immune cells are allogeneic with respect to the individual. 
     
     
         107 . The method of any of  claims 102-105 , wherein the immune cells are autologous with respect to the individual. 
     
     
         108 . The method of any of  claims 101-107 , wherein the individual is human. 
     
     
         109 . The method of any of  claims 101-108 , wherein the individual has cancer. 
     
     
         110 . The method of  claim 109 , wherein the individual has breast cancer, acute myeloid leukemia, multiple myeloma, or glioblastoma. 
     
     
         111 . The method of any of  claims 101-110 , wherein the cells harboring the polynucleotide are administered to the individual once or more than once. 
     
     
         112 . The method of  claim 111 , wherein the duration of time between administrations of the cells harboring the polynucleotide to the individual is 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or one or more years. 
     
     
         113 . The method of any of  claims 101-112 , further comprising the step of providing to the individual an effective amount of an additional therapy. 
     
     
         114 . The method of  claim 113 , wherein the additional therapy comprises surgery, radiation, gene therapy, immunotherapy, or hormone therapy. 
     
     
         115 . The method of any of  claims 101-114 , wherein the cells harboring the polynucleotide are administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, regionally, by perfusion, in a tumor microenvironment, or a combination thereof. 
     
     
         116 . The method of any of  claims 101-115 , further comprising identifying TROP-2-positive cancer in the individual. 
     
     
         117 . The method of any of  claims 101-116 , further comprising producing the cells harboring the polynucleotide. 
     
     
         118 . A polynucleotide that encodes:
 (a) an anti-TROP-2 chimeric antigen receptor (CAR), the CAR comprising (i) an anti-TROP-2 antigen binding region of an RS7 antibody, (ii) a transmembrane domain, and (iii) an intracellular region;   (b) IL-15; and   (c) Caspase 9.   
     
     
         119 . The polynucleotide of  claim 118 , wherein the RS7 antibody is a murine RS7 antibody. 
     
     
         120 . The polynucleotide of  claim 118 , wherein the RS7 antibody is a humanized RS7 antibody. 
     
     
         121 . A polynucleotide that encodes:
 (a) an anti-TROP-2 chimeric antigen receptor (CAR), the CAR comprising (i) an anti-TROP-2 antigen binding region of a 2G10 antibody, (ii) a transmembrane domain, and (iii) an intracellular region;   (b) IL-15; and   (c) Caspase 9.   
     
     
         122 . The polynucleotide of  claim 121 , wherein the 2G10 antibody is a murine 2G10 antibody. 
     
     
         123 . The polynucleotide of  claim 121 , wherein the 2G10 antibody is a humanized 2G10 antibody. 
     
     
         124 . An NK cell comprising the polynucleotide of any of  claims 118-123 . 
     
     
         125 . A natural killer cell comprising:
 (a) a first polynucleotide encoding an anti-TROP-2 chimeric antigen receptor;   (b) a second polynucleotide encoding IL-15; and   (c) a third polynucleotide encoding Caspase 9.   
     
     
         126 . The natural killer cell of  claim 125 , wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are present on the same nucleic acid molecule. 
     
     
         127 . The natural killer cell of  claim 125 , wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are present on two different nucleic acid molecules. 
     
     
         128 . The natural killer cell of  claim 125 , wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are present on three different nucleic acid molecules. 
     
     
         129 . A polynucleotide that encodes for SEQ ID NO:2. 
     
     
         130 . The polynucleotide of  claim 129 , wherein the polynucleotide comprises a sequence having at least 90% identity with SEQ ID NO:1. 
     
     
         131 . The polynucleotide of  claim 130 , wherein the polynucleotide comprises SEQ ID NO:1. 
     
     
         132 . A polynucleotide that encodes for SEQ ID NO:4. 
     
     
         133 . The polynucleotide of  claim 132 , wherein the polynucleotide comprises a sequence having at least 90% identity with SEQ ID NO:3. 
     
     
         134 . The polynucleotide of  claim 133 , wherein the polynucleotide comprises SEQ ID NO:3. 
     
     
         135 . A polynucleotide that encodes for SEQ ID NO:6. 
     
     
         136 . The polynucleotide of  claim 135 , wherein the polynucleotide comprises a sequence having at least 90% identity with SEQ ID NO:5. 
     
     
         137 . The polynucleotide of  claim 136 , wherein the polynucleotide comprises SEQ ID NO:5. 
     
     
         138 . A polynucleotide that encodes for SEQ ID NO:8. 
     
     
         139 . The polynucleotide of  claim 138 , wherein the polynucleotide comprises a sequence having at least 90% identity with SEQ ID NO:7. 
     
     
         140 . The polynucleotide of  claim 139 , wherein the polynucleotide comprises SEQ ID NO:7.